Fluorescence Imaging of Bacterial Killing by Antimicrobial Peptide Dendrimer G3KL

ACS Infect Dis. 2019 Dec 13;5(12):2164-2173. doi: 10.1021/acsinfecdis.9b00299. Epub 2019 Oct 31.

Abstract

We recently discovered that peptide dendrimers such as G3KL ((KL)8(KKL)4(KKL)2KKL, K = branching l-lysine) exert strong activity against Gram-negative bacteria including Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli. Herein, we report a detailed mechanistic study using fluorescence labeled analogs bearing fluorescein (G3KL-Fluo) or dansyl (G3KL-Dansyl), which show a similar bioactivity profile as G3KL. Imaging bacterial killing by super-resolution stimulated emission depletion (STED) microscopy, time-lapse imaging, and transmission electron microscopy (TEM) reveals that the dendrimer localizes at the bacterial membrane, induces membrane depolarization and permeabilization, and destroys the outer leaflet and the inner membrane. G3KL accumulates in bacteria against which it is active; however, it only weakly penetrates into eukaryotic cells without inducing significant toxicity. G3KL furthermore binds to lipopolysaccharide (LPS) and inhibits the LPS induced release of TNF-α by macrophages, similarly to polymyxin B. Taken together, these experiments show that G3KL behaves as a potent membrane disruptive antimicrobial peptide.

Keywords: Pseudomonas aeruginosa; STED microscopy; bacterial membranes; cell penetrating peptides; polymyxin B.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antimicrobial Cationic Peptides / chemistry
  • Antimicrobial Cationic Peptides / pharmacology*
  • Bacterial Outer Membrane / drug effects
  • Cell-Penetrating Peptides / chemistry
  • Cell-Penetrating Peptides / pharmacology*
  • Dendrimers / chemistry*
  • Drug Resistance, Multiple, Bacterial / drug effects
  • Fluorescein / chemistry
  • Fluorescent Dyes / chemistry*
  • Gram-Negative Bacteria / cytology
  • Gram-Negative Bacteria / drug effects
  • Humans
  • Lipopolysaccharides / metabolism
  • Macrophages / drug effects
  • Macrophages / immunology
  • Mice
  • Microscopy, Electron, Transmission
  • Molecular Structure
  • Phosphatidylcholines / chemistry
  • RAW 264.7 Cells
  • Time-Lapse Imaging
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • 1-myristoyl-2-(12-((5-dimethylamino-1-naphthalenesulfonyl)amino)dodecanoyl)-sn-glycero-3-phosphocholine
  • Antimicrobial Cationic Peptides
  • Cell-Penetrating Peptides
  • Dendrimers
  • Fluorescent Dyes
  • Lipopolysaccharides
  • Phosphatidylcholines
  • Tumor Necrosis Factor-alpha
  • Fluorescein